The field of the invention relates to methods for screening for the binding affinity of chemical entities to other bioactive molecules and identifying covalent inhibitors of bioactive molecules. In particular, the field of the invention relates to methods for screening for the binding affinity of small molecule inhibitors of bioactive molecules such as enzymes that contain catalytic or non-catalytic cysteines including the neuronal precursor cell-expressed developmentally down-regulated 4-1 ubiquitin ligase (NEDD4-1), and pharmaceutical compositions and therapeutic methods that include or utilize the screened small molecule inhibitors.
Fragment based drug discovery (FBDD) has emerged as a powerful approach to discover drug leads by exploring greater chemical diversity space with smaller libraries.1 The major challenge, however, is to detect weak binding interactions between drug-like fragments and their protein target. Disulfide tethering was developed as one solution to this problem.2 In this approach, disulfide-containing fragments are covalently trapped on the protein surface via the reversible formation of disulfide bonds. Subsequent MS of the intact protein can identify the covalently bound fragment. The advantages of this method include screening the fragments as mixtures rather than as separate entities. Screening fragments as mixtures increases the throughput capability of the assay and reduces the number of false positives by introducing competition between the fragments. This has proven to be a general and successful approach.3 Another technique relies on the use of an α-cyanoacrylamide moiety attached to drug-like fragments that react reversibly with non-catalytic cysteines present at the binding site of the protein of interest.4 
Whether it is possible to design a robust system where the protein can select the best binder from a mixture of electrophilic fragments under irreversible conditions to identify novel leads is not known. Such an approach would be particularly powerful since the identified fragments can subsequently retain their electrophilic tether while being elaborated into a covalent drug. Irreversible tethering would especially benefit the burgeoning field of covalent drug-discovery.5 
However, one concern with such an approach is the danger of selecting the most reactive fragment rather than the fragment with the most specific binding affinity to the protein target.6 If the electrophilic fragments are too reactive, cysteines or other nucleophilic residues present on the protein surface can undergo non-specific covalent modifications by the fragments irrespective of their binding affinity.7 Alternatively hyper-reactive cysteines or other nucleophilic residues can nonspecifically react with even moderately electrophilic fragments, leading to non-specific covalent modifications of the protein.8 In addition, no systematic studies have been done to investigate the kinetic reactivity of cysteine reactive electrophiles attached to a large number (˜50) drug-like fragments, in order to outline general principles and design rules for irreversible tethering. While this work was in progress Nonoo, et. al. reported the first irreversible tethering method using a small ten-member acrylamide library, which included known reversible thymidylate synthase inhibitor scaffolds.9 However, hyper-reactive acrylamide in that library has led to one false positive hit, and no systematic studies have been done further to investigate the reactivity of and outline design rules for drug-like libraries for irreversible tethering. Moreover, there are still no reports of irreversible fragment screening of an unbiased library to identify novel and selective binding fragments. Therefore, whether it is possible to rationally design an electrophilic library of drug-like fragments for irreversible tethering is still a concern.
Here, the inventors address this concern and shows that the proper selection of a cysteine reactive electrophile yields a chemical system that can select weakly bound electrophilic fragments from a mixture, and covalently trap the best binders at the highly reactive catalytic cysteine of the model cysteine protease papain. The discovered fragments behave as weak and irreversible inhibitors of papain, and have novel non-peptidic structures. The reported method serves as an entry point to discover non-peptidic inhibitors of other cysteine proteases, which are promising drug targets to treat parasitic infections.10 
NEDD4-1 is overexpressed in a wide variety of cancers and is a promising drug target for these diseases. It is proposed that NEDD4-1 ubiquitinate and degrade tumor suppressors p53, LATS, and PTEN, which contributes to its oncogenic properties. Genetic experiments have firmly established the essential role of Nedd4-1 in regulating insulin and IGF-1 growth pathways in cells, two pathways that are upregulated in many human cancers such as Ewing's sarcoma (Nat. Struct. Mol. Biol. 2014 June; 21(6):522-7). Furthermore, Nedd4-1−/+ heterozygous mice gained less weight when placed on the high fat diet, which suggests that Nedd4-1 is a potential drug target to treat obesity (Endocrinology. 2015 April; 156(4):1283-91). In addition NEDD4-1 is involved in the degradation of α-synuclein, a key player in Parkinson'S disease, which makes Nedd4-1 a promising drug target to treat parkinson's disease. Therefore, small molecule activators of NEDD4-1 will serve as therapeutics to treat Parkinson's disease. Lastly, NEDD4-1 and its closely related homolog NEDD4-2, which is also a likely target of our inhibitors, have been shown to be essential host proteins for the budding of HIV and Ebola viruses from the host cell (J. Virol. 2014 July; 88(13):7294-306). Therefore, NEDD4 inhibitors are promising host targets to treat HIV.
Unlike protein kinases, efforts to develop small molecule inhibitors of ubiquitin ligases have been mostly unsuccessful. Here, the inventors disclose small molecule inhibitors of the HFECT-type ubiquitin ligase NEDD4-1, an enzyme for which there are no reported inhibitors. These compounds serve as therapies for the multitude of diseases in which NEDD4-1 is a factor including, but not limited to, cancer, neurodegenerative disease and spreading of HIV.